Patent Application
20150284793
The ASCII file, entitled 62147SequenceListing.txt, created on Apr. 2, 2015, comprising 112,931 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.
The present invention, in some embodiments thereof, relates to methods and kits for diagnosing schizophrenia.
Currently diagnosis of schizophrenia relies solely on the analysis of a person's symptoms. Diagnosis is made from information obtained from physical examination, taking a person's family history and emotional history, as well as a medical evaluation, and a mental status examination. Relying on symptomatic history makes diagnosis of schizophrenia difficult, particularly since no single symptom is definitive for diagnosis. Rather, the diagnosis encompasses a pattern of signs and symptoms, in conjunction with impaired occupational or social functioning. Currently diagnosis includes looking for delusions (false beliefs strongly held in spite of invalidating evidence); visual, auditory, tactile, olfactory or gustatory hallucinations; disorganized speech; disorganized thinking; grossly disorganized thinking and/or catatonic behavior; negative symptoms, such as emotional deficit, avolition (inability to initiate and persist in goal-directed activities) and alogia (poverty of speech) are also symptoms of schizophrenia. Continuous signs of the disturbance must persist for at least 6 months. This 6-month period must include at least 1 month of active-phase symptoms (listed above) (or less if successfully treated) and may include periods of prodromal or residual symptoms. During these prodromal or residual periods, the signs of the disturbance may be manifested by only negative symptoms or two or more active-phase symptoms in an attenuated form (e.g., odd beliefs, unusual perceptual experiences).
Diagnosis of schizophrenia is made even harder because it is often difficult to differentiate schizophrenia from other mental disorders including bipolar disorder, schizoaffective disorder, and brief psychotic disorder. In addition, diagnosis of schizophrenia is often confused with other organic medical conditions (e.g. encephalitis) or substance conditions (drugs of abuse, such as amphetamines and phencyclidine, or other medications). Although recently brain imaging techniques have been utilized as a tool towards diagnosis, this is costly, is inconvenient to patients, and is not considered very reliable.
Eukaryotic RNA transcripts can undergo a range of post-transcriptional modifications, which increase the diversity of the transcriptome without requiring increases in genome size. These include alternative splicing and RNA editing. RNA editing refers to post-transcriptional processes that alter the nucleotide sequence of an RNA transcript by insertion, deletion or nucleotide conversion. In mammals, the most prevalent form of RNA editing involves the conversion of adenosine to inosine (A-to-I) by hydrolytic deamination at the C6 position of adenine. A-to-I editing, which is catalyzed by enzymes of the adenosine deaminase acting on RNA (ADAR) family, is most prevalent in the central nervous system (CNS) but occurs in many tissues. Once an adenosine nucleotide is converted to an inosine, it acts in a manner similar to a guanosine nucleotide, with a number of potential consequences. When this conversion occurs in the coding region of mRNA, it results in an altered nucleotide codon and, therefore, can change the amino acid sequence of the coded protein in what is referred to as a re-coding editing event. A-to-I editing can also result in the creation or elimination of splice sites, potentially altering the portions of the RNA that remain in the final product. Additionally, the A-to-I conversion alters base pairing, because inosine pairs preferentially with cytidine, and this potentially affects the secondary structure of the RNA. In the case of RNA molecules that bind target RNA segments, such as microRNAs (miRNAs), the altered base pairing can change binding specificities. Thus, A-to-I editing in both translated and untranslated regions of RNA can be biologically significant.
The best studied A-to-I RNA editing event accrues in the AMPA glutamate receptor subunit GluR2 Q/R site. Virtually 100% of the transcripts of this gene are edited at this site such that the mRNA contains an arginine (R) codon (CIG) in place of the genomic glutamate (Q) codon (CAG). Underediting of the GluR2 Q/R Q/R site greatly increases the Ca2+ permeability of AMPA receptors. The increase in Ca2+ influx through the receptor channel may cause neural cell death. Heterozygous mice, carriers of a modified GluR2 which can not be edited, show increased AMPAR Ca2+ permeability causing epileptic seizures and premature death. In 2004 Kawahara and his colleagues published a study showing a defect in the RNA editing of the glutamate receptor in ALS patients [Nature, Vol. 427, February 2004]. They found that the editing efficiency varied between 0% and 100% in the motor neurons from each individual with ALS, and was incomplete in 56% of them. All the control motor neurons derived from healthy patients examined showed 100% editing efficiency. When they examined the editing efficiency in Purkinji cells (non-affected cells) from these patients they saw no difference between the ALS patients and the normal group.
Until recently, only a handful of A-to-I editing sites were known in the human transcriptome. However, several years ago, it was revealed that the extent of editing is much larger, affecting tens of thousands of sites and more than 1,600 different genes.
Using an inosine-specific cleavage reaction, Morse et al. [Proc. Natl. Acad. Sci. U.S.A. 99(2002) 7906-7911] conducted a targeted search for additional A-to-I substitutions and revealed clusters of editing sites in 19 human brain derived mRNAs. Of the clusters, 15 out of 19 occurred in repetitive elements, mainly in Alu sequences, within non-coding sequences. In addition, three independent groups performed systematic searches using computational algorithms that corroborated the existence and extent of abundant A-to-I editing modifications, mainly in Alu repetitive elements in non-coding regions, such as introns and untranslated regions [E. Y. Levanon, et al., Nat. Biotechnol. 22 (2004), 1001-1005; Athanasiadis et al., PLoS Biol. 2004 December; 2(12); D. D. Kim et al., Genome Res. 14 (2004) 1719-1725].
Background art includes Slotkin et al., Genome Med. 2013; 5:105. doi: 10.1186/gm508; Niswender Cmet al., 2001; 5:478-491. doi: 10.1016/S0893-133X(00)00223-2; and Dracheva S, et al Mol Psychiatry. 2007; 5:1001-1010.
Additional background art includes International Application WO 2005087949 and WO2011031786.
According to an aspect of some embodiments of the present invention there is provided a method of diagnosing schizophrenia, the method comprising analyzing in a biological sample of a subject a level of A-to-I RNA editing of at least one CNS-expressed gene as set forth in Table 1, wherein an amount of the A-to-I RNA editing of the at least one gene below a predetermined level is indicative of schizophrenia in the subject.
According to an aspect of some embodiments of the present invention there is provided a method of treating a subject suspected of having schizophrenia, the method comprising:
(a) diagnosing a subject with schizophrenia according to claim 1; and
(b) treating the subject according to the results of the diagnosing.
According to an aspect of some embodiments of the present invention there is provided a method of monitoring treatment of a subject having schizophrenia, the method comprising:
(a) providing the subject with a treatment for the schizophrenia;
(b) analyzing in a sample of the subject a level of A-to-I RNA editing in at least one CNS-expressed gene set forth in Table 1, wherein an increase in the A-to-I RNA editing of the gene compared to the level of A-to I RNA editing of the gene prior to the providing is indicative of a therapeutic treatment.
According to an aspect of some embodiments of the present invention there is provided a kit comprising a first primer set for amplifying one of the CNS-expressed genes set forth in Table 1 and a second primer set for amplifying a second of the CNS-expressed genes set forth in Table 1.
According to an aspect of some embodiments of the present invention there is provided a kit comprising at least two oligonucleotides, wherein the first of the at least two oligonucleotides hybridizes to a first sequence of cDNA of a first gene set forth in Table 1, and a second of the at least two oligonucleotides hybridizes to a second sequence of cDNA of a second gene set forth in Table 1, wherein the first and the second sequence are differentially A-to I edited in a schizophrenia subject as compared with a non-schizophrenia subject.
According to some embodiments of the invention, the analyzing is effected on the polynucleotide level.
According to some embodiments of the invention, the analyzing is effected on the polypeptide level.
According to some embodiments of the invention, the analyzing is effected by sequencing a portion of the at least one CNS-expressed gene that comprises the A-to-I RNA editing site.
According to some embodiments of the invention, the biological sample is selected from the group consisting of blood, serum, CSF, saliva, mucosal sample and a cortical brain sample.
According to some embodiments of the invention, the blood comprises peripheral blood nucleated cells.
According to some embodiments of the invention, the analyzing is effected using oligonucleotides specific to sites of the RNA editing.
According to some embodiments of the invention, the analyzing is effected by:
(a) amplifying a portion of the at least one CNS-expressed gene that comprises the A-to-I RNA editing site; and
(b) sequencing the portion of the at least one CNS-expressed gene.
According to some embodiments of the invention, the kit further comprises agents for sequencing the first and the second CNS expressed genes.
According to some embodiments of the invention, the kit further comprises a reverse transcriptase enzyme.
According to some embodiments of the invention, the kit is for diagnosing schizophrenia.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
The present invention, in some embodiments thereof, relates to methods and kits for diagnosing schizophrenia.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
A-to-I RNA editing is a post-transcriptional modification that converts adenosines to inosines in both coding and noncoding RNA transcripts. It is catalyzed by ADAR (adenosine deaminase acting on RNA) enzymes, which exist throughout the body but are most prevalent in the central nervous system. Inosines exhibit properties that are most similar to those of guanosines. As a result, ADAR-mediated editing can post-transcriptionally alter codons, introduce or remove splice sites, or affect the base pairing of the RNA molecule with itself or with other RNAs. A-to-I editing is a mechanism that regulates and diversifies the transcriptome. Although altered A to I editing patterns have been found in epileptic mice, suicide victims suffering chronic depression and in malignant gliomas, the full biological significance of ADARs is not understood.
The present inventors have now found that biological samples derived from schizophrenic patients show a statistically significant decrease in the levels of A-to-I editing in particular CNS-expressed genes—see Table 1 of the Examples section herein below. The present inventors propose that analysis of A-to-I editing of these genes can serve as a basis for diagnosing this disease. This may be effected by looking at particular A-I editing sites on the genes and/or by looking at the overall A-I editing of the entire gene.
Thus, according to a first aspect of the present invention there is provided a method of diagnosing schizophrenia, the method comprising analyzing in a biological sample of a subject a level of A-to-I RNA editing of at least one CNS-expressed gene as set forth in Table 1, wherein an amount of the A-to-I RNA editing of the at least one gene below a predetermined level is indicative of schizophrenia in the subject.
As used herein, the term “diagnosing” refers to determining the presence of a disease, classifying a disease, determining a severity of the disease (grade or stage), monitoring disease progression, forecasting an outcome of the disease and/or prospects of recovery.
The term “schizophrenia” or “SCZ” as used herein may be used to refer to the SCZ-spectrum disorders, Schizotypal Personality Disorder (SPD) and Schizoaffective Disorder (SD), as well as Schizophrenia under the narrower, DSM-IV definition and even to affective psychoses.
The conversion of adenosine to inosine (A-to-I) in RNA editing is brought about by hydrolytic deamination at the C6 position of adenine. A-to-I editing is catalyzed by enzymes of the adenosine deaminase acting on RNA (ADAR) family. Once an adenosine nucleotide is converted to an inosine, it acts in a manner similar to a guanosine nucleotide (i.e. base-pairing with cytosine). Three primary members of the ADAR family have been identified in humans: ADAR1, ADAR2 and ADAR3. These proteins are highly conserved across vertebrates. ADAR1 is expressed in both the constitutive p110 isoform and the interferon-inducible p150 isoform. ADAR1 and ADAR2 are present in many tissues, whereas ADAR3 is specifically expressed in brain tissues. ADARs contain a conserved deaminase domain that mediates A-to-I editing, as well as variable double-stranded RNA-binding domains that are required for substrate specificity and binding. Homodimerization of ADARs is required for editing activities, as observed in vitro and confirmed with in vivo studies.
The biological sample of this aspect of the present invention may be derived from brain tissue (for example cell extracts) or may be derived from a fluid of the subject. Thus, the present invention contemplates analyzing blood, serum, plasma, blood cells, urine, sputum, saliva, stool, spinal fluid or CSF, lymph fluid, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears or milk of the subject.
Brain tissue samples are typically obtained by a surgical procedure, for example during a biopsy.
According to one embodiment, a sample of blood is obtained from a subject according to methods well known in the art. In some embodiments, a drop of blood is collected from a simple pin prick made in the skin of a subject. Blood may be drawn from a subject from any part of the body (e.g., a finger, a hand, a wrist, an arm, a leg, a foot, an ankle, a stomach, and a neck) using techniques known to one of skill in the art, in particular methods of phlebotomy known in the art.
The amount of blood collected will vary depending upon the site of collection, the amount required for a method of the invention, and the comfort of the subject. However, an advantage of one embodiment of the present invention is that the amount of blood required to implement the methods of the present invention can be so small that more invasive procedures are not required to obtain the sample. For example, in some embodiments, all that is required is a drop of blood. This drop of blood can be obtained, for example, from a simple pinprick. In various specific embodiments, 0.001 ml, 0.005 ml, 0.01 ml, 0.05 ml, 0.1 ml, 0.15 ml, 0.2 ml, 0.25 ml, 0.5 ml, 0.75 ml, 1 ml, 1.5 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 15 ml or more of blood is collected from a subject. In another embodiment, 0.001 ml to 15 ml, 0.01 ml to 10 ml, 0.1 ml to 10 ml, 0.1 ml to 5 ml, 1 to 5 ml of blood is collected from a subject.
In some embodiments of the present invention, blood is stored within a K3/EDTA tube. In another embodiment, one can utilize tubes for storing blood which contain stabilizing agents such as disclosed in U.S. Pat. No. 6,617,170 (which is incorporated herein by reference). In another embodiment the PAXgene™ blood RNA system: provided by PreAnalytiX, a Qiagen/BD company may be used to collect blood. In yet another embodiment, the Tempus™ blood RNA collection tubes, offered by Applied Biosystems may be used. Tempus™ collection tubes provide a closed evacuated plastic tube containing RNA stabilizing reagent for whole blood collection.
The blood collected is preferably utilized immediately or within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours or is optionally stored at temperatures such as 4° C., or at −20° C. prior to use in accordance with the methods of the invention. In some embodiments, a portion of the blood sample is used in accordance with the invention at a first instance of time whereas one or more remaining portions of the blood sample (or fractions thereof) are stored for a period of time for later use. For longer term storage, storage methods well known in the art, such as storage at cryo temperatures (e.g. below −60° C. can be used. In some embodiments, in addition to storage of the blood or instead of storage of the blood, plasma, serum, isolated nucleic acid or proteins are stored for a period of time for later use in accordance with methods known in the art.
In one aspect, whole blood is obtained from an individual according to the methods of phlebotomy well known in the art. Whole blood includes blood which can be used directly, and includes blood wherein the serum or plasma has been removed and the RNA or mRNA from the remaining blood sample has been isolated in accordance with methods well known in the art (e.g., using, preferably, gentle centrifugation at 300 to 800.times.g for 5 to 10 minutes). In a specific embodiment, whole blood (i.e., unfractionated blood) obtained from a subject is mixed with lysing buffer (e.g., Lysis Buffer (1 L): 0.6 g EDTA; 1.0 g KHCO.sub.2, 8.2 g NH.sub.4C1 adjusted to pH 7.4 (using NaOH)), the sample is centrifuged and the cell pellet retained, and RNA or mRNA extracted in accordance with methods known in the art (“lysed blood”) (see for example Sambrook et al.). The use of unfractionated whole blood is preferred since it avoids the costly and time-consuming process to separate out the cell types within the blood (Kimoto, 1998, Mol. Gen. Genet 258:233-239; Chelly J et al., 1989, Proc. Nat. Acad. Sci. USA 86:2617-2621; Chelly J et al., 1988, Nature 333:858-860).
In some embodiments of the present invention, whole blood collected from a subject is fractionated (i.e., separated into components). In specific embodiments of the present invention, blood cells are separated from whole blood collected from a subject using techniques known in the art. For example, blood collected from a subject can be subjected to Ficoll-Hypaque (Pharmacia) gradient centrifugation. Such centrifugation separates erythrocytes (red blood cells) from various types of nucleated cells and from plasma. In particular, Ficoll-Hypaque gradient centrifugation is useful to isolate peripheral blood leukocytes (PBLs) which can be used in accordance with the methods of the invention.
According to one embodiment, identifying the level of A to I editing in an expressed gene is effected on the polynucleotide level.
Quantitating the amount of A to I editing may be effected over the entire length of the gene such that the average A to I editing of a particular gene is determined. Alternatively, the amount of A to I editing at a single or a combination of editing sites may be determined.
The amount of A to I editing may be compared to a control sample derived from a patient that does not have schizophrenia (e.g. a healthy subject). Alternatively, the amount of A to I editing may be compared to reference amounts known to be present in healthy subjects.
According to one embodiment, downregulation of A to I editing typically refers to a decrease by at least 5%, 10%, 12%, 15%, 17% 20% or greater of A to I in at least one editing site of at least one gene which appears in Table 1.
According to one embodiment, downregulation of A to I editing typically refers to a decrease by at least 5%, 10%, 12%, 15%, 17% 20% or greater of A to I in at least two editing site of the at least one gene.
According to one embodiment, downregulation of A to I editing typically refers to a decrease by at least 5%, 10%, 12%, 15%, 17% 20% or greater of A to I in at least three editing site of the at least one gene.
According to one embodiment, downregulation of A to I editing typically refers to a decrease by at least 5%, 10%, 12%, 15%, 17% 20% or greater of A to I in at least four editing site of the at least one gene.
According to one embodiment, downregulation of A to I editing typically refers to a decrease by at least 5%, 10%, 12%, 15%, 17% 20% or greater of A to I in at least five editing site of the at least one gene.
According to one embodiment, downregulation of A to I editing typically refers to a decrease by at least 5%, 10%, 12%, 15%, 17% 20% or greater of A to I in the majority of editing sites of the at least one gene.
According to one embodiment, downregulation of A to I editing typically refers to a decrease by at least 5%, 10%, 12%, 15%, 17% 20% or greater of A to I in all the editing site of the at least one gene.
According to one embodiment, downregulation of A to I editing typically refers to a decrease by at least 5%, 10%, 12%, 15%, 17% 20% or greater of A to I in at least one editing site of at least two genes which appears in Table 1.
According to one embodiment, downregulation of A to I editing typically refers to a decrease by at least 5%, 10%, 12%, 15%, 17% 20% or greater of A to I in at least one editing site of at least five genes which appears in Table 1.
According to one embodiment, downregulation of A to I editing typically refers to a decrease by at least 5%, 10%, 12%, 15%, 17% 20% or greater of A to I in at least one editing site of at least ten genes which appears in Table 1.
According to one embodiment, downregulation of A to I editing typically refers to a decrease by at least 5%, 10%, 12%, 15%, 17% 20% or greater of A to I in at least one editing site of all the genes which appears in Table 1.
According to one embodiment, the analysis of A to I editing is effected on the polynucleotide level.
The RNA of a sample is typically isolated and the amount of times an A to I editing event takes place on the RNA.
Isolation, extraction or derivation of RNA may be carried out by any suitable method. Isolating RNA from a biological sample generally includes treating a biological sample in such a manner that the RNA present in the sample is extracted and made available for analysis. Any isolation method that results in extracted RNA may be used in the practice of the present invention. It will be understood that the particular method used to extract RNA will depend on the nature of the source.
Preferably, RNA is isolated from a biological sample (e.g. blood) by the following protocol. Lysis Buffer is added to blood sample in a ratio of 3 parts Lysis Buffer to 1 part blood (Lysis Buffer (1 L) 0.6 g EDTA; 1.0 g KHCO2, 8.2 g NH4Cl adjusted to pH 7.4 (using NaOH)). Sample is mixed and placed on ice for 5-10 minutes until transparent. Lysed sample is centrifuged at 1000 rpm for 10 minutes at 4° C., and supernatant is aspirated. Pellet is resuspended in 5 ml Lysis Buffer, and centrifuged again at 1000 rpm for 10 minutes at 4° C. Pelleted cells are homogenized using TRIzol® (GIBCO/BRL) in a ratio of approximately 6 ml of TRIzol® for every 10 ml of the original blood sample and vortexed well. Samples are left for 5 minutes at room temperature. RNA is extracted using 1.2 ml of chloroform per 1 ml of TRIzol®. Sample is centrifuged at 12,000 g for 5 minutes at 4° C. and upper layer is collected. To upper layer, isopropanol is added in ratio of 0.5 ml per 1 ml of TRIzol®. Sample is left overnight at −20° C. or for one hour at −20° C. RNA is pelleted in accordance with known methods, RNA pellet air dried, and pellet resuspended in DEPC treated ddH2O. RNA samples can also be stored in 75% ethanol where the samples are stable at room temperature for transportation.
Purity and integrity of RNA can be assessed by absorbance at 260/280 nm and agarose gel electrophoresis followed by inspection under ultraviolet light. Preferably RNA integrity is assessed using more sensitive techniques such as the Agilent 2100 Bioanalyzer 6000 RNA Nano Chip.
The sample may be processed before the method is carried out, for example RNA purification may be carried out following the extraction procedure. Processing of the sample may involve one or more of: filtration, distillation, centrifugation, extraction, concentration, dilution, purification, inactivation of interfering components, addition of reagents, and the like.
Analysis of the editing events may be effected on the RNA molecules themselves present in the sample or on cDNA which has been reverse transcribed from the RNA in the sample.
Reverse transcription is achieved by forming a reaction mixture comprising the sample RNA, at least one primer capable of hybridizing to the RNA, a reverse transcriptase, and deoxynucleoside triphosphates (dNTPs) to produce cDNA. Reverse transcription of the inosine “I” residue results in a guanine “G” residue in the corresponding cDNA. Reverse transcription of the adenosine “A” residue results in a thymine “T” residue in the corresponding cDNA.
As used herein, “reverse transcriptase” generally refers to an enzyme capable of replicating RNA into a complementary DNA (cDNA). Reverse transcription is the process of copying an RNA template into DNA. In some embodiments, a reverse transcriptase is an enzyme capable of creating a DNA strand using an RNA strand as a template for synthesis. In one example, the enzyme optimally has the reverse transcriptase activity to generate a DNA from an RNA template, wherein the enzyme either does not have a DNA polymerase activity or has a minimal DNA polymerase activity. In another example, the enzyme has nominal DNA polymerase activity and high reverse transcriptase activity. Either a reverse transcriptase or a DNA polymerase with reverse transcriptase activity may generate a cDNA strand from RNA template. The reverse transcriptase may be a naturally occurring reverse transcriptase enzyme, or a variant or fragment thereof that retains the desired enzymatic activity described above. Any recombinantly engineered reverse transcriptase enzyme produced by routine methods in the field of molecular biology that has reverse transcriptase activity may be used in the practice of the present invention.
The term “reverse transcriptase primer” or “RT primer” as used herein (also known as a cDNA primer) refers to an oligonucleotide capable of acting as a point of initiation of cDNA synthesis by an RT under suitable conditions. Thus, a reverse transcription reaction is primed by an RT primer. The appropriate length of an RT primer typically ranges from 6 to 50 nucleotides or from 15 to 35 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the mRNA template, but may still be used. A primer need not reflect the exact sequence of the template nucleic acid, but must be sufficiently complementary to hybridize with the template. The design of suitable primers for cDNA synthesis is well known in the art.
Optionally, the cDNA (or portion thereof) may be amplified prior to analysis using a PCR reaction. Typically, the portion of the cDNA which is amplified comprises the A to I editing site that the present inventors have identified. A typical length of an amplicon is about 100-300 by (for example about 200). Please provide range of lengths of amplicons contemplated. The location of the editing sites in each gene is provided in Table 2 of the Examples section. Thus, by way of example the primers used to amplify the portion of CACNA1D cDNA should flank position 53820892 etc.
Exemplary primer pairs are provided in Table 2, herein below.
According to a specific embodiment, the amplification primer/s is labeled with a bar-code (i.e. identification sequence). The barcode sequence is useful during multiplex reactions when a number of samples are pooled in a single reaction. The barcode sequence may be used to identify a particular molecule, sample or library. The barcode sequence may be between 3-400 nucleotides, more preferably between 3-200 and even more preferably between 3-100 nucleotides. Thus, the barcode sequence may be 6 nucleotides, 7 nucleotides, 8, nucleotides, nine nucleotides or ten nucleotides.
The primers may include additional sequences that are necessary for a sequencing process in a downstream reaction, as further described herein below.
According to one embodiment, the method of this aspect of the present invention is carried out using an isolated oligonucleotide which hybridizes to either the A-I edited variant or the non-edited variant by complementary base-pairing in a sequence specific manner, and is capable of distinguishing between the two variants. Oligonucleotides typically comprises a region of complementary nucleotide sequence that hybridizes under stringent conditions to at least about 8, 10, 13, 16, 18, 20, 22, 25, 30, 40, 50, 55, 60, 65, 70, 80, 90, 100, 120 (or any other number in-between) or more consecutive nucleotides in a target nucleic acid molecule. Depending on the particular assay, the consecutive nucleotides can either include the A-I editing site nucleic acid sequence, or be a specific region in close enough proximity 5′ and/or 3′ to the editing site nucleic acid sequence to carry out the desired assay.
According to one embodiment, the oligonucleotide is a probe. The probe may hybridize to the A to I edited site to provide a detectable signal under experimental conditions and not hybridize to the non-edited site to provide a detectable signal under identical experimental conditions. Alternatively, the probe may hybridize to the A to I non-edited site to provide a detectable signal under experimental conditions and not hybridize to the edited site to provide a detectable signal under identical experimental conditions.
The probes of this embodiment of this aspect of the present invention may be, for example, affixed to a solid support (e.g., arrays or beads).
According to another embodiment, the oligonucleotide is a primer of a primer pair. As used herein, the term “primer” refers to an oligonucleotide which acts as a point of initiation of a template-directed synthesis using methods such as PCR (polymerase chain reaction) or LCR (ligase chain reaction) under appropriate conditions (e.g., in the presence of four different nucleotide triphosphates and a polymerization agent, such as DNA polymerase, RNA polymerase or reverse-transcriptase, DNA ligase, etc, in an appropriate buffer solution containing any necessary co-factors and at suitable temperature(s)). Such a template directed synthesis is also called “primer extension”. For example, a primer pair may be designed to amplify a region of DNA using PCR. Such a pair will include a “forward primer” and a “reverse primer” that hybridize to complementary strands of a DNA molecule and that delimit a region to be synthesized/amplified. A primer of this aspect of the present invention is capable of amplifying, together with its pair (e.g. by PCR) an A-I edited site nucleic acid sequence to provide a detectable signal under experimental conditions and which does not amplify the non-edited site to provide a detectable signal under identical experimental conditions or vice versa.
According to additional embodiments, the oligonucleotide is about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. While the maximal length of a probe can be as long as the target sequence to be detected, depending on the type of assay in which it is employed, it is typically less than about 50, 60, 65, or 70 nucleotides in length. In the case of a primer, it is typically less than about 30 nucleotides in length. In a specific preferred embodiment of the invention, a primer or a probe is within the length of about 18 and about 28 nucleotides. It will be appreciated that when attached to a solid support, the probe may be of about 30-70, 75, 80, 90, 100, or more nucleotides in length.
The oligonucleotide of this aspect of the present invention need not reflect the exact sequence of the A to I edited site nucleic acid sequence (i.e. need not be fully complementary), but must be sufficiently complementary to hybridize with the A to I edited site sequence under the particular experimental conditions. Accordingly, the sequence of the oligonucleotide typically has at least 70% homology, preferably at least 80%, 90%, 95%, 97%, 99% or 100% homology, for example over a region of at least 13 or more contiguous nucleotides with the target nucleic acid sequence. The conditions are selected such that hybridization of the oligonucleotide to the edited or non-edited sites favored and hybridization to the non-edited or edited site.
By way of example, hybridization of short nucleic acids (below 200 by in length, e.g. 13-50 by in length) can be effected by the following hybridization protocols depending on the desired stringency; (i) hybridization solution of 6×SSC and 1% SDS or 3 M TMACl, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 0.1% nonfat dried milk, hybridization temperature of 1-1.5° C. below the Tm, final wash solution of 3 M TMACl, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5° C. below the Tm (stringent hybridization conditions) (ii) hybridization solution of 6×SSC and 0.1% SDS or 3 M TMACl, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 0.1% nonfat dried milk, hybridization temperature of 2-2.5° C. below the Tm, final wash solution of 3 M TMACl, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5° C. below the Tm, final wash solution of 6×SSC, and final wash at 22° C. (stringent to moderate hybridization conditions); and (iii) hybridization solution of 6×SSC and 1% SDS or 3 M TMACl, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 0.1% nonfat dried milk, hybridization temperature at 2.5-3° C. below the Tm and final wash solution of 6×SSC at 22° C. (moderate hybridization solution).
Various considerations must be taken into account when selecting the stringency of the hybridization conditions. For example, the more closely the oligonucleotide reflects a sequence that is present in the A to I editing site, the higher the stringency of the assay conditions should be, although the stringency must not be too high so as to prevent hybridization of the oligonucleotides to the target sequence. Further, the lower the homology of the oligonucleotide to the editing site nucleic acid sequence, the lower the stringency of the assay conditions should be, although the stringency must not be too low to allow hybridization to non specific nucleic acid sequences.
Oligonucleotides of the invention may be prepared by any of a variety of methods (see, for example, J. Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 1989, 2.sup.nd Ed., Cold Spring Harbour Laboratory Press: New York, N.Y.; “PCR Protocols: A Guide to Methods and Applications”, 1990, M. A. Innis (Ed.), Academic Press: New York, N.Y.; P. Tijssen “Hybridization with Nucleic Acid Probes—Laboratory Techniques in Biochemistry and Molecular Biology (Parts I and II)”, 1993, Elsevier Science; “PCR Strategies”, 1995, M. A. Innis (Ed.), Academic Press: New York, N.Y.; and “Short Protocols in Molecular Biology”, 2002, F. M. Ausubel (Ed.), 5.sup.th Ed., John Wiley & Sons: Secaucus, N.J.). For example, oligonucleotides may be prepared using any of a variety of chemical techniques well-known in the art, including, for example, chemical synthesis and polymerization based on a template as described, for example, in S. A. Narang et al., Meth. Enzymol. 1979, 68: 90-98; E. L. Brown et al., Meth. Enzymol. 1979, 68: 109-151; E. S. Belousov et al., Nucleic Acids Res. 1997, 25: 3440-3444; D. Guschin et al., Anal. Biochem. 1997, 250: 203-211; M. J. Blommers et al., Biochemistry, 1994, 33: 7886-7896; and K. Frenkel et al., Free Radic. Biol. Med. 1995, 19: 373-380; and U.S. Pat. No. 4,458,066.
For example, oligonucleotides may be prepared using an automated, solid-phase procedure based on the phosphoramidite approach. In such a method, each nucleotide is individually added to the 5′-end of the growing oligonucleotide chain, which is attached at the 3′-end to a solid support. The added nucleotides are in the form of trivalent 3′-phosphoramidites that are protected from polymerization by a dimethoxytriyl (or DMT) group at the 5′-position. After base-induced phosphoramidite coupling, mild oxidation to give a pentavalent phosphotriester intermediate and DMT removal provides a new site for oligonucleotide elongation. The oligonucleotides are then cleaved off the solid support, and the phosphodiester and exocyclic amino groups are deprotected with ammonium hydroxide. These syntheses may be performed on oligo synthesizers such as those commercially available from Perkin Elmer/Applied Biosystems, Inc. (Foster City, Calif.), DuPont (Wilmington, Del.) or Milligen (Bedford, Mass.). Alternatively, oligonucleotides can be custom made and ordered from a variety of commercial sources well-known in the art, including, for example, the Midland Certified Reagent Company (Midland, Tex.), ExpressGen, Inc. (Chicago, Ill.), Operon Technologies, Inc. (Huntsville, Ala.), and many others.
Purification of the oligonucleotides of the invention, where necessary or desirable, may be carried out by any of a variety of methods well-known in the art. Purification of oligonucleotides is typically performed either by native acrylamide gel electrophoresis, by anion-exchange HPLC as described, for example, by J. D. Pearson and F. E. Regnier (J. Chrom., 1983, 255: 137-149) or by reverse phase HPLC (G. D. McFarland and P. N. Borer, Nucleic Acids Res., 1979, 7: 1067-1080).
The sequence of oligonucleotides can be verified using any suitable sequencing method including, but not limited to, chemical degradation (A. M. Maxam and W. Gilbert, Methods of Enzymology, 1980, 65: 499-560), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (U. Pieles et al., Nucleic Acids Res., 1993, 21: 3191-3196), mass spectrometry following a combination of alkaline phosphatase and exonuclease digestions (H. Wu and H. Aboleneen, Anal. Biochem., 2001, 290: 347-352), and the like.
As already mentioned above, modified oligonucleotides may be prepared using any of several means known in the art. Non-limiting examples of such modifications include methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc), or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc). Oligonucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc), intercalators (e.g., acridine, psoralen, etc), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc), and alkylators. The oligonucleotide may also be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the oligonucleotide sequences of the present invention may also be modified with a label.
In certain embodiments, the detection probes or amplification primers or both probes and primers are labeled with a detectable agent or moiety before being used in amplification/detection assays. In certain embodiments, the detection probes are labeled with a detectable agent. Preferably, a detectable agent is selected such that it generates a signal which can be measured and whose intensity is related (e.g., proportional) to the amount of amplification products in the sample being analyzed.
The association between the oligonucleotide and detectable agent can be covalent or non-covalent. Labeled detection probes can be prepared by incorporation of or conjugation to a detectable moiety. Labels can be attached directly to the nucleic acid sequence or indirectly (e.g., through a linker). Linkers or spacer arms of various lengths are known in the art and are commercially available, and can be selected to reduce steric hindrance, or to confer other useful or desired properties to the resulting labeled molecules (see, for example, E. S. Mansfield et al., Mol. Cell. Probes, 1995, 9: 145-156).
Methods for labeling nucleic acid molecules are well-known in the art. For a review of labeling protocols, label detection techniques, and recent developments in the field, see, for example, L. J. Kricka, Ann. Clin. Biochem. 2002, 39: 114-129; R. P. van Gijlswijk et al., Expert Rev. Mol. Diagn. 2001, 1: 81-91; and S. Joos et al., J. Biotechnol. 1994, 35: 135-153. Standard nucleic acid labeling methods include: incorporation of radioactive agents, direct attachments of fluorescent dyes (L. M. Smith et al., Nucl. Acids Res., 1985, 13: 2399-2412) or of enzymes (B. A. Connoly and O. Rider, Nucl. Acids. Res., 1985, 13: 4485-4502); chemical modifications of nucleic acid molecules making them detectable immunochemically or by other affinity reactions (T. R. Broker et al., Nucl. Acids Res. 1978, 5: 363-384; E. A. Bayer et al., Methods of Biochem. Analysis, 1980, 26: 1-45; R. Langer et al., Proc. Natl. Acad. Sci. USA, 1981, 78: 6633-6637; R. W. Richardson et al., Nucl. Acids Res. 1983, 11: 6167-6184; D. J. Brigati et al., Virol. 1983, 126: 32-50; P. Tchen et al., Proc. Natl. Acad. Sci. USA, 1984, 81: 3466-3470; J. E. Landegent et al., Exp. Cell Res. 1984, 15: 61-72; and A. H. Hopman et al., Exp. Cell Res. 1987, 169: 357-368); and enzyme-mediated labeling methods, such as random priming, nick translation, PCR and tailing with terminal transferase (for a review on enzymatic labeling, see, for example, J. Temsamani and S. Agrawal, Mol. Biotechnol. 1996, 5: 223-232). More recently developed nucleic acid labeling systems include, but are not limited to: ULS (Universal Linkage System), which is based on the reaction of mono-reactive cisplatin derivatives with the N7 position of guanine moieties in DNA (R. J. Heetebrij et al., Cytogenet. Cell. Genet. 1999, 87: 47-52), psoralen-biotin, which intercalates into nucleic acids and upon UV irradiation becomes covalently bonded to the nucleotide bases (C. Levenson et al., Methods Enzymol. 1990, 184: 577-583; and C. Pfannschmidt et al., Nucleic Acids Res. 1996, 24: 1702-1709), photoreactive azido derivatives (C. Neves et al., Bioconjugate Chem. 2000, 11: 51-55), and DNA alkylating agents (M. G. Sebestyen et al., Nat. Biotechnol. 1998, 16: 568-576).
Any of a wide variety of detectable agents can be used in the practice of the present invention. Suitable detectable agents include, but are not limited to, various ligands, radionuclides (such as, for example, .sup.32P, .sup.35S, .sup.3H, .sup.14C, .sup.125I, .sup.131I, and the like); fluorescent dyes (for specific exemplary fluorescent dyes, see below); chemiluminescent agents (such as, for example, acridinium esters, stabilized dioxetanes, and the like); spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper and platinum) or nanoclusters; enzymes (such as, for example, those used in an ELISA, i.e., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase); colorimetric labels (such as, for example, dyes, colloidal gold, and the like); magnetic labels (such as, for example, Dynabeads™); and biotin, dioxigenin or other haptens and proteins for which antisera or monoclonal antibodies are available.
In certain embodiments, the inventive detection probes are fluorescently labeled. Numerous known fluorescent labeling moieties of a wide variety of chemical structures and physical characteristics are suitable for use in the practice of this invention. Suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxy-fluorescein, 6 carboxyfluorescein or FAM), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine or TMR), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin and aminomethylcoumarin or AMCA), Oregon Green Dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514), Texas Red, Texas Red-X, Spectrum Red™, Spectrum Green™, cyanine dyes (e.g., Cy-3™, Cy-5™, Cy-3.5™, Cy-5.5™), Alexa Fluor dyes (e.g., Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), IRDyes (e.g., IRD40, IRD 700, IRD 800), and the like. For more examples of suitable fluorescent dyes and methods for linking or incorporating fluorescent dyes to nucleic acid molecules see, for example, “The Handbook of Fluorescent Probes and Research Products”, 9th Ed., Molecular Probes, Inc., Eugene, Oreg. Fluorescent dyes as well as labeling kits are commercially available from, for example, Amersham Biosciences, Inc. (Piscataway, N.J.), Molecular Probes Inc. (Eugene, Oreg.), and New England Biolabs Inc. (Beverly, Mass.).
As mentioned, identification of the editing site may be carried out using an amplification reaction.
As used herein, the term “amplification” refers to a process that increases the representation of a population of specific nucleic acid sequences in a sample by producing multiple (i.e., at least 2) copies of the desired sequences. Methods for nucleic acid amplification are known in the art and include, but are not limited to, polymerase chain reaction (PCR) and ligase chain reaction (LCR). In a typical PCR amplification reaction, a nucleic acid sequence of interest is often amplified at least fifty thousand fold in amount over its amount in the starting sample. A “copy” or “amplicon” does not necessarily mean perfect sequence complementarity or identity to the template sequence. For example, copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable but not complementary to the template), and/or sequence errors that occur during amplification.
A typical amplification reaction is carried out by contacting a forward and reverse primer (a primer pair) to the sample DNA together with any additional amplification reaction reagents under conditions which allow amplification of the target sequence.
The terms “forward primer” and “forward amplification primer” are used herein interchangeably, and refer to a primer that hybridizes (or anneals) to the target (template strand). The terms “reverse primer” and “reverse amplification primer” are used herein interchangeably, and refer to a primer that hybridizes (or anneals) to the complementary target strand. The forward primer hybridizes with the target sequence 5′ with respect to the reverse primer.
The term “amplification conditions”, as used herein, refers to conditions that promote annealing and/or extension of primer sequences. Such conditions are well-known in the art and depend on the amplification method selected. Thus, for example, in a PCR reaction, amplification conditions generally comprise thermal cycling, i.e., cycling of the reaction mixture between two or more temperatures. In isothermal amplification reactions, amplification occurs without thermal cycling although an initial temperature increase may be required to initiate the reaction. Amplification conditions encompass all reaction conditions including, but not limited to, temperature and temperature cycling, buffer, salt, ionic strength, and pH, and the like.
As used herein, the term “amplification reaction reagents”, refers to reagents used in nucleic acid amplification reactions and may include, but are not limited to, buffers, reagents, enzymes having reverse transcriptase and/or polymerase activity or exonuclease activity, enzyme cofactors such as magnesium or manganese, salts, nicotinamide adenine dinuclease (NAD) and deoxynucleoside triphosphates (dNTPs), such as deoxyadenosine triphospate, deoxyguanosine triphosphate, deoxycytidine triphosphate and thymidine triphosphate. Amplification reaction reagents may readily be selected by one skilled in the art depending on the amplification method used.
According to this aspect of the present invention, the amplifying may be effected using techniques such as polymerase chain reaction (PCR), which includes, but is not limited to Allele-specific PCR, Assembly PCR or Polymerase Cycling Assembly (PCA), Asymmetric PCR, Helicase-dependent amplification, Hot-start PCR, Intersequence-specific PCR (ISSR), Inverse PCR, Ligation-mediated PCR, Methylation-specific PCR (MSP), Miniprimer PCR, Multiplex Ligation-dependent Probe Amplification, Multiplex-PCR, Nested PCR, Overlap-extension PCR, Quantitative PCR (Q-PCR), Reverse Transcription PCR (RT-PCR), Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), Bridge PCR (primers are covalently linked to a solid-support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR (where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thermal ‘step’ to favour solid support priming), Thermal asymmetric interlaced PCR (TAIL-PCR), Touchdown PCR (Step-down PCR), PAN-AC and Universal Fast Walking.
According to another embodiment, the amount of A-I editing at a particular site is analyzed by sequencing the cDNA and analyzing the proportion of molecules that comprise the edited sequence: non-edited sequence.
Methods for sequence determination are generally known to the person skilled in the art. Preferred are next generation sequencing methods or parallel high throughput sequencing methods. An example of an envisaged sequence method is pyrosequencing, in particular 454 pyrosequencing, e.g. based on the Roche 454 Genome Sequencer. This method amplifies DNA inside water droplets in an oil solution with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony. Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs. Yet another envisaged example is Illumina or Solexa sequencing, e.g. by using the Illumina Genome Analyzer technology, which is based on reversible dye-terminators. DNA molecules are typically attached to primers on a slide and amplified so that local clonal colonies are formed. Subsequently one type of nucleotide at a time may be added, and non-incorporated nucleotides are washed away. Subsequently, images of the fluorescently labeled nucleotides may be taken and the dye is chemically removed from the DNA, allowing a next cycle. Yet another example is the use of Applied Biosystems' SOLiD technology, which employs sequencing by ligation. This method is based on the use of a pool of all possible oligonucleotides of a fixed length, which are labeled according to the sequenced position. Such oligonucleotides are annealed and ligated. Subsequently, the preferential ligation by DNA ligase for matching sequences typically results in a signal informative of the nucleotide at that position. Since the DNA is typically amplified by emulsion PCR, the resulting bead, each containing only copies of the same DNA molecule, can be deposited on a glass slide resulting in sequences of quantities and lengths comparable to Illumina sequencing. A further method is based on Helicos' Heliscope technology, wherein fragments are captured by polyT oligomers tethered to an array. At each sequencing cycle, polymerase and single fluorescently labeled nucleotides are added and the array is imaged. The fluorescent tag is subsequently removed and the cycle is repeated. Further examples of sequencing techniques encompassed within the methods of the present invention are sequencing by hybridization, sequencing by use of nanopores, microscopy-based sequencing techniques, microfluidic Sanger sequencing, or microchip-based sequencing methods. The present invention also envisages further developments of these techniques, e.g. further improvements of the accuracy of the sequence determination, or the time needed for the determination of the genomic sequence of an organism etc.
According to one embodiment, the sequencing method comprises deep sequencing.
As used herein, the term “deep sequencing” and variations thereof refers to the number of times a nucleotide is read during the sequencing process. Deep sequencing indicates that the coverage, or depth, of the process is many times larger than the length of the sequence under study.
It will be appreciated that any of the analytical methods described herein can be embodied in many forms. For example, it can be embodied in on a tangible medium such as a computer for performing the method operations. It can be embodied on a computer readable medium, comprising computer readable instructions for carrying out the method operations. It can also be embodied in electronic device having digital computer capabilities arranged to run the computer program on the tangible medium or execute the instruction on a computer readable medium.
Computer programs implementing the analytical method of the present embodiments can commonly be distributed to users on a distribution medium such as, but not limited to, CD-ROMs or flash memory media. From the distribution medium, the computer programs can be copied to a hard disk or a similar intermediate storage medium. In some embodiments of the present invention, computer programs implementing the method of the present embodiments can be distributed to users by allowing the user to download the programs from a remote location, via a communication network, e.g., the internet. The computer programs can be run by loading the computer instructions either from their distribution medium or their intermediate storage medium into the execution memory of the computer, configuring the computer to act in accordance with the method of this invention. All these operations are well-known to those skilled in the art of computer systems.
As mentioned, A to I editing in the coding region of mRNA, may result in an altered nucleotide codon and, therefore, the amino acid sequence of the coded protein may be altered during A to I editing.
Therefore, the present inventors contemplate analyzing the level of A to I editing using an antibody which is capable of selectively binding to an epitope of one of the variants and not the other. As used herein, the term “epitope” refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
The term “antibody” as used in this invention includes intact molecules as well as functional fragments thereof (such as Fab, F(ab′)2, Fv, scFv, dsFv, or single domain molecules such as VH and VL) that are capable of binding to an epitope of an antigen.
Suitable antibody fragments for practicing some embodiments of the invention include a complementarity-determining region (CDR) of an immunoglobulin light chain (referred to herein as “light chain”), a complementarity-determining region of an immunoglobulin heavy chain (referred to herein as “heavy chain”), a variable region of a light chain, a variable region of a heavy chain, a light chain, a heavy chain, an Fd fragment, and antibody fragments comprising essentially whole variable regions of both light and heavy chains such as an Fv, a single chain Fv Fv (scFv), a disulfide-stabilized Fv (dsFv), an Fab, an Fab′, and an F(ab′)2.
As used herein, the terms “complementarity-determining region” or “CDR” are used interchangeably to refer to the antigen binding regions found within the variable region of the heavy and light chain polypeptides. Generally, antibodies comprise three CDRs in each of the VH (CDR HI or HI; CDR H2 or H2; and CDR H3 or H3) and three in each of the VL (CDR LI or LI; CDR L2 or L2; and CDR L3 or L3).
The identity of the amino acid residues in a particular antibody that make up a variable region or a CDR can be determined using methods well known in the art and include methods such as sequence variability as defined by Kabat et al. (See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.), location of the structural loop regions as defined by Chothia et al. (see, e.g., Chothia et al., Nature 342:877-883, 1989.), a compromise between Kabat and Chothia using Oxford Molecular's AbM antibody modeling software (now Accelrys®, see, Martin et al., 1989, Proc. Nati Acad Sci USA. 86:9268; and world wide web sitebioinf-orgdotuk/dabs), available complex crystal structures as defined by the contact definition (see MacCallum et al., J. Mol. Biol. 262:737-745, 1996) and the “conformational definition” (see, e.g., Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008).
As used herein, the “variable regions” and “CDRs” may refer to variable regions and CDRs defined by any approach known in the art, including combinations of approaches.
Kits
Any of the components described herein may be comprised in a kit. In a non-limiting example the kit comprises at least two primer pairs, each primer pair for amplifying a cDNA sequence of one of the genes set forth in Table 2, wherein the amplification product comprises an A-I editing site set forth in Table 2, herein below, each component being in a suitable container.
In another non-limiting example, the kit comprises at least two oligonucleotides, the first oligonucleotide hybridizing to the cDNA of a first gene set forth in Table 1, and a second oligonucleotide hybridizing to the cDNA of a second gene set forth in Table 1. The sequences to which the oligonucleotides hybridize are differentially A-to I edited in a schizophrenia subject as compared with a non-schizophrenia subject. According to one embodiment, the oligonucleotides are labeled with a detectable moiety as further described herein above. Preferably the two oligonucleotides are labeled with different detectable moieties, so that it is possible to determine the amount of hybridization of each of the oligonucleotides individually.
Additional components that may be included in the kit include: a reverse transcriptase and optionally reagents for additional reactions such as: (i) a polydT oligonucleotide; (ii) a DNA polymerase; (iii) MgCl2; and/or (iv) RNAse H. The kit may also comprise reaction components for sequencing the amplified sequences.
As mentioned, herein above the primers may also comprise a barcoding sequence and additional sequences which aid in downstream sequencing reactions.
The containers of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other containers, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a container.
When the components of the kit are provided in one or more liquid solutions, the liquid solution can be an aqueous solution. However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent.
A kit will preferably include instructions for employing, the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented.
Once a diagnosis has been formed according to the methods described herein, further corroboration of the diagnosis may be effected. Specifically, the “International Classification of Diseases” (ICD) of the World Health Organization (WHO), and the “Diagnostic and Statistical Manual of Mental Disorders” (DSM) of the American Psychiatric Association (APA) can be used as the diagnostic criteria for schizophrenia.
In addition, once a diagnosis has been formed according to methods described herein, a treatment agent regiment/dosage may be recommended. Examples of therapeutics which may be recommended include, for example, Aripiprazole, Clozapine, ziprasidone, respiradone, quetiapine or olanzapine.
The methods described herein may also be useful for monitoring a therapeutic (e.g. agent or treatment).
Thus, according to another aspect of the present invention there is provided a method of monitoring treatment of a subject having schizophrenia, the method comprising:
(a) providing the subject with a treatment for the schizophrenia;
(b) analyzing in a sample of the subject a level of A-to-I RNA editing in at least one CNS-expressed gene set forth in Table 1, wherein an increase in the A-to-I RNA editing of the gene compared to the level of A-to I RNA editing of the gene prior to the providing is indicative of a therapeutic treatment.
Examples of therapeutic agents have been provided herein above.
As used herein the term “about” refers to ±10%.
The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
The term “consisting of” means “including and limited to”.
The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.
The aim of the present study was to evaluate the difference in RNA editing levels in brain samples taken from schizophrenia samples compared to controls at specific editing sites in the CNS, and explore the association of RNA editing with schizophrenia as well as the possibility to use it as a biomarker.
Materials and Methods
Brain Samples:
Cortical brain tissue (BA10) from post mortem of schizophrenia patients (n=20) and controls (n=20) were obtained.
Tissue Processing:
RNA was extracted using TRI reagent according to manufacturer's instructions. Thereafter, cDNA samples were prepared from 2 μg of Dnase I-treated total RNA using a mix of random hexamers and oligo dT from the Bio-Rad advanced iScript kit with compliance to the manufacturer instructions. 200 ng of 1st strand cDNA were loaded on the Access-Array micro-fluidic device for the analysis of RNA editing.
Experimental System:
The experimental system applied in this study was set to detect the discrepancy between DNA sequence and its corresponding RNA sequence, like in the cases of A-to-I RNA editing. In order to enhance throughput, and to ensure the uniform amplification of multiple transcripts which enables a more accurate quantification of A-to-G ratios within these transcripts, an assay was developed that couples microfluidics-based PCR and next generation sequencing [Li, J. B., Science 324, 1210-1213].
DNA Sequencing and Bioinformatic Sequence Analysis:
The UCSC genome browser Human February 2009 (GRCh37/hg19) Assembly was used for identifying any discrepancies between the Refseq data to that obtained from the actual DNA sequencing output. For the screen, a targeted-resequencing approach of NGS (next generation sequencing) was used to generate and sequence multiple PCR amplicons containing the target editing site/s. The analysis of data obtained, was performed to detect any A/G mismatches within the cDNA sequences. Such mismatches were summed and scored for their signal strength according to the overall number of coverage reads and more important to the percentage of A-to-G levels.
Targeted Re-Sequencing of RNA Editing Sites in RNA Samples Using the Fluidigm Access Array Coupled with the Ion-Torrent PGM:
To precisely detect and measure the levels of A-to-I RNA editing in wild type and mutant samples, targeted amplicons were generated and barcoded using a two-step PCR strategy which also minimized the total number of primers required. The target gene specific primers were designed using Primer 3.0 [http://frododotwidotmitdotedu/] to be located in exons while spanning introns, thus avoiding DNA contaminations to the RNA and by the 454 tool for designing of fusion primers supplemented with universal consensus sequences.
The Fluidigm Access Array is a high-throughput target-enrichment system designed to produce PCR products that could be compatible with all of the major next-generation sequencers. It enables the creation of enriched multiple PCR products from 48 samples, all at once. Using the Access Array IFC one can automatically assemble 2,304 PCR reactions, each reaction combining cDNA from one of the 48 samples and one of the 48 primer pairs. The FLDGM-AA amplification and tagging strategy is based on two consecutive PCR reactions, each done with specific fusion PCR primers. The first PCR is performed “on chip” and generates amplicons containing the editing target sites flanked by common universal sequences [CS1 (fused to the forward primer)/CS2 (fused to the reverse primer)]. The second PCR is performed “off chip”, on a thermal cycler and uses the first PCR's products as templates. The CS regions previously conjoined (by the previous PCR) enables the attachment of various barcodes to the amplicons, generating longer products. These longer amplicons contains not only the 10 bps sample specific barcode sequences, as well as the Ion-Torrent PGM tr-P1 & A-seq adaptors. Thus, the final PCR output per each sample is a mini-library that is consisted of multiple sequences representing the 48 different target specific primers-pairs, whereas all of them are tagged with the same barcode sequence, which is representative of a single RNA sample. Accordingly, the unified library that is loaded for sequencing is comprised of all 48 mini-libraries represents the entire samples panel.
A schematic representation of the three major steps in the quantification of multiple RNA editing sites by next generation sequencing is presented in
Amplification of the Target Regions Containing the Target Editing Sites Using the Fluidigm Access Array Microfluidic System:
4 μl of single primers-pair (4 μM per primer in 1× AA-loading buffer) were loaded into the primer inlets of the 48.48 Access Array IFC (Fluidigm). To prepare the cDNA templates, we added 2.25 μl of each cDNA sample to 2.75 μl of pre-sample mix containing the following enzyme and reagents from the Roche FastStart High Fidelity PCR System; 0.5 μl of 10× FastStart High Fidelity Reaction Buffer wo/Mg, 0.5 μl DMSO [5%], 0.1 μl 10 mM PCR Grade Nucleotide Mix [200 μM], 0.9 μl 25 mM MgCl2 [4.5 Mm], 0.25 μl 20× Access Array Loading Reagent (Fluidigm), 0.05 μl of FastStart High Fidelity Enzyme Blend and 0.7 μl of PCR grade water. 4 μl of this mix were loaded into the samples inlets of the 48.48 Access Array IFC (Fluidigm). After the loading of both samples and primers via IFC Controller AX (Fluidigm) loading script, the IFC was subject to thermal cycling using FC1 Cycler (Fluidigm) with the following program for 40 cycles: 50° C. for 2:00 min. 70° C. for 20:00 min. 95° C. 10 minutes. 10 cycles of: 95° C. for 15 sec; 59.5° C. for 30 sec; 72° C. for 1 min; 4 cycles of: 95° C. for 15 sec; 80° C. for 30 sec; 59.5° C. for 30 sec; 72° C. for 1 min; 10 cycles of: 95° C. for 15 sec; 59.5° C. for 30 sec; 72° C. for 1 min; 4 cycles of: 95° C. for 15 sec; 80° C. for 30 sec; 60° C. for 30 sec; 72° C. for 1 min; 8 cycles of: 95° C. for 15 sec; 59.5° C. for 30 sec; 72° C. for 1 min; 4 cycles of: 95° C. for 15 sec; 80° C. for 30 sec; 60° C. for 30 sec; 72° C. for 1 min; Finalizing with 72° C. for 3 min. Once PCR has terminated, the IFC was transferred to another IFC Controller AX (Fluidigm) and mini-libraries were harvested by the controller harvest script.
Sequencing Adaptor and Barcode Addition:
For each sample, 1.0 μl of the PCR products harvested from the IFC was 1:110 diluted and added to 15 μl of pre-sample mix containing the following enzyme and reagents from the Roche FastStart High Fidelity PCR System; 2 μl of 10× FastStart High Fidelity Reaction Buffer wo/Mg, 1 μl DMSO [5%], 0.4 μl 10 mM PCR Grade Nucleotide Mix [200 μM], 3.6 μl 25 mM MgCl2 [4.5 mM], 0.2 μl of FastStart High Fidelity Enzyme Blend and 7.8 μl of PCR grade water. To that samples mix, 4 μl of primer mix from the 2 μM Access Array Barcode Library for Ion Torrent PGM Sequencer—96 (P/N100-4911), utilizing the B-set; A-BC-CS2 and P1-CS1 barcode primer combination. We used the following PCR program: 95° C. for 10 min; 10 cycles of 95° C. for 30 s, 60° C. for 30 s and 72° C. for 1 min; and 72° C. for 5 min.
Fluidigm Library Sequencing Data Analysis:
Libraries were pooled and sequenced on Ion-Torrent PGM using the Ion PGM™ Sequencing 200 Kit v2 and the 1G-Ion 318™ Chip Kit v2 (Life Technologies, Grand Island, N.Y. 14072, USA).
Pre-Alignment Processing:
The sequencing data was downloaded from the machine as fastq file. First, all raw sequences data was de-indexed into 48 samples according to the barcodes used by an in-house script. All reads were trimmed of the universal CS1 and CS2 sequences and all short reads (<20 nts) were removed. Alignment of the processed reads was made using bwa version 0.7.4-r385, using the mem option and the parameters: -k 20 -B 3 -O 3 -T 20, for seed in the length of the average primer, and for considering the Ion typical error of small indels.
Alignment Process:
The alignment was done to the human refseq data base, where reads that were aligned to more than one location were omitted from further analysis. Samtools mpileup was used on the alignment results and in-house script was run to move the results to the genomic locations from the refseqs and then an in-house script to count the number of different nucleotides in each genomic location that had a q-score≧20. The last stage was to filter the results to a preset set of locations of interest, for each location we present the total number of reads which had good quality per each sample, and the calculated percentage of reads that have a ‘G’ at the specified genomic location [#of ‘G’ reads/(#of ‘G’ reads+#of ‘A’ reads)].
Editing Levels Calling:
After establishing the percentage of editing, the present inventors next turned to call on the validated novel RNA-editing sites. For that they required that each editing-sites variant will have coverage of at least 300 reads and to be supported by at least six mismatch reads (≧2%) with base quality score ≧20 and mapping quality score ≧20. All known SNPs present in dbSNP (UCSC; Common SNPs (135)) were removed.
Statistical Analysis:
T-test was conducted for the comparison between editing levels in schizophrenia samples compared to controls in each of the RNA editing sites, followed by the Benjamini-Hochberg procedure for multiple testing. Statistical significance was considered for corrected P<0.05.
Results
A general decrease in the levels of A-to-I RNA editing in schizophrenia patients was observed compared to controls. Significantly decreased editing was found in as many as 25 out of the 103 editing sites that were evaluated. Some of these sites are located on genes that encode for neurotransmitter receptors. These include GABRA3 (GABA receptor subunit alpha-3 precursor), exhibiting 11.49% decrease in RNA editing in schizophrenia samples compared to control (P=0.029). Three editing sites at the GRIA2 gene (AMPA 2 glutamate receptor 2) were noted, in one a 28.99% decrease (P<0.001) was observed. GRIA4 displayed 27.92% decrease (P=0.005). Two editing sites located on the GRIK2 gene (kainate2 glutamate receptor) were noted, in one a 24.96% decrease was observed (P<0.001). Interestingly, a 28.9% decrease in editing in the HTR2C gene (Serotonin receptor 2C) in schizophrenia samples (P=0.006) was noted. The present inventors also observed a decrease in two editing sites in schizophrenia samples on genes that encode calcium (21.65%, P=0.001) and potassium (26.55%, P=0.003) voltage gated ion channels.
Genes in which RNA editing is significantly decreased in schizophrenia samples as compared to healthy samples are set forth in Table 1, herein above.
Detected editing levels are represented in percentage as manifestations of the ratio calculated by between sequences reads that contain a ‘G’ to reads contains an ‘A’ according to the following formula [(#G/(#A-F#G)*100].
The overall calculation of the mean editing levels of all 25 genes in healthy and in SCZ samples shows a significant difference in editing (
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.
This application claims the benefit of priority under 35 USC 119(e) of U.S. Provisional Patent Application No. 61/974,588 filed Apr. 3, 2014, the contents of which are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61974588 | Apr 2014 | US |
